Although malignant hyperthermia is associated primarily with humans and was first realized as a reaction to inhalation anaesthetics, it is understood that MH is also a very common problem in certain animals, particularly pigs. There is therefore particular commercial interest in developing suitable assays to determine MH in pigs, as well as providing suitable diagnosis to test humans to avoid life threatening circumstances in the operating room.
Malignant hyperthermia (MH) is an inherited predisposition to a hypermetabolic syndrome (adverse reaction) triggered by inhalation anaesthetics such as halothane and some skeletal muscle relaxants such as succinylcholine. The primary defect in MH is related to a sustained increase in myoplasmic calcium which causes muscle contracture and increased glycolysis concomitant with the production of H.sub.2 O, CO.sub.2 and heat and excessive consumption of O.sub.2. Other signs of the disorder (including the hyperthermia for which it was named) may be explained as a direct result of muscle contracture and increased glycolysis [Steward, D. J. and O'Connor, G. A. R, "Malignant Hyperthermia--The Acute Crisis", in Britt B. A. ed. Malignant Hyperthermia, Boston, Martinus Nijhoff, (1987)].
The disease in humans is a serious health problem as the affected individuals are usually unaware of their condition and problem with a potentially lethal reaction to the drugs administered at surgery. The observed frequency of the disease is dependent on the drugs administered. The highest estimate based on the use of succinylcholine in combination with halothane, gives a frequency of 1 in 4200 anaesthetics [Ording, H., Anesth Analg. 64: 700-704, (1985)]. This may be a gross underestimate of the true gene frequency since many individuals who carry the gene are never exposed to the triggering agent and thus remain undiagnosed. Additionally the incidence of masseter muscle spasm following halothane-succinylcholine induction of anaesthesia is 1 in 200; 50% of these individuals have been subsequently shown to have biopsies positive for MH suggesting that the true incidence of the trait may be considerably higher [Rosenberg, H, and Fletcher, J. E., Anesth. Analg. 65: 161-164, (1986)]. In many families the disease segregates as an autosomal dominant condition [Kalow, W., "Inheritance of Malignant Hyperthermia--A review of Published Data", in Britt, B. A. Ed. Malignant Hyperthermia, supra, (1987)] although other modes of inheritance have been reported in some families.
The mortality rate for MH in North America has decreased from 84% in the 1960s to about 7% [Britt, B. A., "Preface: A History of Malignant Hyperthermia", in Britt B. A. ed. Malignant Hyperthermia, Boston, Martinus Nijhoff, pp 1-10, (1987)], following improvements in monitoring systems, increased awareness of MH and the advent of dantrolene treatment in 1975. A marked elevation in end-tidal (exhaled) carbon dioxide levels is an early indicator of an MH reaction and,where monitored and recognized, may allow prompt treatment with sodium dantrolene (dantrium) to avert a full crisis. The fatality rate is still unacceptably high in many countries in the world ( e.g. 24% in the U.K.).
Fatalities may result from one or more of multiple complications in a fulminant MH crisis. Skeletal muscle, smooth (involuntary) muscle and cardiac muscle are all affected in an MH reaction. Contraction of smooth muscle of the blood vessels causes hypertension which further decreases the oxygen supply and results in accelerated deep breathing. Pulmonary edema may occur as the crisis progresses especially at the onset of cardiac failure. Cardiac failure is triggered both by rigidity of the heart muscle and by elevated levels of potassium in the blood. Once the temperature of an affected individual has begun to rise it does so rapidly (1.degree. C. every five minutes) and final temperatures as high as 46.degree. C. have been reported. Leakage of myoglobin into the blood as a result of membrane damage may trigger kidney failure in survivors. Some survivors never regain consciousness and others have central nervous system damage (e.g. paralysis, blindness, deafness, impaired intelligence, speech defects) as a result of extremely high fever and/or electrolyte imbalance [Steward D. J. and O'Connor, G. A. R. supra, (1987)].
Accurate laboratory tests are required which can detect individuals at risk for developing malignant hyperthermia. Currently, the best test for individuals at risk is a diagnostic muscle biopsy. This test, described by Kalow et al. ["Metabolic error of muscle metabolism after recovery from malignant hyperthermia", Lancet, 2: 895-898, (1970)], is based on the abnormal contracture response of MH muscle to caffeine, halothane and a combination of the two. It is a highly invasive procedure, requiring 10-15 grams of thigh muscle. Moreover the tests are time-consuming and sometimes inconclusive. The concordance between tests is poor such that an individual may be labelled "at risk" by the criteria of one test and "not at risk" by the criteria of a second. Other individuals may be equivocal due to overlap in the values for "at risk" and "not at risk" groups. Control values and diagnostic "cut-off" points have to be established in each laboratory so that it is difficult to establish new units to test for MH susceptibility. Moreover, such an invasive technique is inappropriate for general population screening prior to anaesthesia. Thus, the availability of a DNA based diagnostic test is of major significance and utility for detection of individuals at risk for malignant hyperthermia.
Porcine halothane sensitivity represents an excellent animal model for malignant hyperthermia. The clinical crisis in pigs follows a very similar course to that in humans and crises may be similarly arrested or averted by prompt treatment with dantrolene sodium. In pigs, however, the syndrome may additionally be triggered by over-exercise and/or stress. Usually over-exercise is not a significant problem in the raising of pigs. However, stress is a problem as particularly experienced during shipping and prior to slaughter. The pig industry loses hundreds of thousands of dollars a year due to deaths or spoiled meat caused by pigs being susceptible to malignant hyperthermia.
While it has been described as a recessive condition in pigs, it is more likely to be a co-dominant condition since muscle biopsy studies [Britt, B. A., et al, "Malignant Hyperthermia--pattern of inheritance in Swine". In Aldrete J. A. eds., Second International Symposium on Malignant Hyperthermia. New York, Grune and Stratton, pp 195-211, (1978)] reveal that heterozygous pigs may be mildly affected and homozygous MH/MH pigs may be more seriously affected.
The gene responsible for halothane sensitivity (HAL) has been found to segregate in pigs with a number of other genetic markers including S (S Locus affecting expression of A-O red blood antigens), Phi (glucose phosphate isomerase), H (H locus encoding blood group antigens), Po2 (postalbumin-2) and PgD (6-phospho-gluconate dehydrogenase), [Archibald, A. L. and Imlah, P., Animal Blood Groups and Biochem. Genet. 16: 253-263, (1985)]. It is therefore assumed that these genes are linked on one pig chromosome. Since genetic linkage groups are often conserved throughout the animal kingdom and since the human equivalents of three of these genetic markers (Phi, Po2 and H) have been found to map to human chromosome 19 [Shaw, D. and Eiberg, H., Report of the Committee for chromosomes 17, 18, and 19. Human Gene Mapping 9 (1987); Ninth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet, vol 46, Nos. 1-4, (1987)], there was reason to suggest that the human gene for MH may also be in this gene cluster on chromosome 19. A possible further localization of MH to the long arm (q) of chromosome 19 was suggested by the fact that the H gene analog maps to 19q and the Phi gene analog (GPI) maps to band 19q12-19q13 on 19q (Shaw and Eiberg, supra).
In recent years it has been possible to track genetic disease genes in families using closely linked genetic markers. The most commonly used marker of a chromosome site is a restriction enzyme cleavage site that may be present (+) on the pair of chromosomes in some members of the population or absent (-) at the same site in the chromosome pair of other members of the population. Still other individuals will be heterozygous (.+-.) having one chromosome of each type [Botstein, E. et al, Am. J. Hum. Genet. 32: 314-331, (1980)]. The (+) chromosome can be distinguished from the (-) chromosome by extracting DNA from the blood cells (or other cells) of the test individual, treating it with the restriction enzyme whose cleavage site is "polymorphic" (i.e. cleaves or doesn't cleave) and fractionating the DNA fragments by size. The DNA from a chromosome without the cleavage site gives a larger fragment than the DNA from a chromosome with the cleavage site, providing an assay for the presence or absence of the cleavage site. The term RFLP is an acronym for restriction fragment length polymorphism. An RFLP constitutes a genetic marker allowing the polymorphic chromosome site to be tracked in a family. Any genetic disease that tracks (segregates) in a family with the RFLP marker is considered to be linked to the marker, that is it maps near to the marker on the chromosome which they share.